The pancreatic acinar cell has been studies in our lab for many years as a model for examining the intracellular transport pathway in a non- neuroendocrine regulated secretor) cell. Recent data from our laboratory indicate that the pancreatic acinar cell contains several immunoreactive members of the SNARE complex of secretory vesicle and target plasma membrane proteins discovered in yeast and neuronal cells. Several of these may be novel mammalian homologues and include isoforms of v-SNAREs (cellubrevin), tSNAREs (syntaxin), and proteins that control membrane fusion (Rab3D, synaptotagmin). This information provides the basis for future studies in which we will examine the role of these proteins in this nonneuroendocrine regulated secretory cell. The Specific Aims are: First, we will characterize membrane proteins involved in regulated exocytosis along the following lines: 1. We will complete the characterization of Rab3D, the acinar cell low Mr GTP binding protein that is granule-specific and likely serves as a molecular switch for regulated exocytosis; 2. We will pursue the molecular characterization of v- and t-SNAREs and associated proteins (synaptotagmin) in the acinar cell and their co-association in compartments along the secretory pathway. This will be carried out by molecular cloning of cDNAs and immunogold immunocytochemistry. 3. We will determine if SNARE proteins relocated in the cell following intense secretagogue stimulation that is associated with amplification of the Golgi: and 4. We plan to assess the function of SNARE proteins in the acinar cell using permeabilized cells which allow entry of probes (antibodies against SNAREs etc. and botulinal toxins). They will be analyzed for effects on the secretory pathway including the function of cellubrevin in maturation of regulated and constitutive secretory vesicles and on exocytosis. Second, we will examine the molecular mechanisms of membrane fusion on the secretory pathway using cell free assays for zymogen granule/plasma membrane fusion and formation of constitutive secretory vesicles. This will allow us to test directly under controlled conditions the effects of antibodies against SNAREs, synaptotagmin, Rab3D and other proteins implicated in membrane targeting and fusion. Third, exocytosis from the acinar cell is associated with massive relocation of secretory granule membranes to the apical surface which is compensated for by membrane retrieval, likely to the Golgi complex. We will examine the route and kinetics of retrieval from stimulated pancreatic lobules using immunogold electron microscopy and antibodies against membrane components of the exocytic and endocytic compartments. Immunoisolation of recycling vesicles from acinar cells stimulated by secretagogues in vitro should allow us to identify associations of membrane proteins during compensatory membrane retrieval. Particular attention will be paid to formation of coated vesicles and the relationship of synaptotagmin and proteins of the SNARE complex in membrane recycling.